Apparatus for the detection of pepsin

ABSTRACT

An apparatus for detecting pepsin comprising a solid support, and a peptide chain wherein the peptide chain is operatively configured to be cleaved by pepsin, and the peptide chain is disposed on a surface of the solid support.

FIELD OF THE INVENTION

This invention relates, in one embodiment, to medical diagnostictesting, and more particularly to a medical test useful for thedetection of enzymes, and in particular pepsin.

BACKGROUND OF THE INVENTION

Gastroesophageal reflux disease (GERD) represents a host of symptoms andpathologies believed to result from the inappropriate exposure of theesophagus and upper airway to gastric acid. A wide host of diseases areattributed to this exposure, ranging from esophageal injury to the widehost of disorders termed “laryngopharyngeal reflux”.

As is disclosed in U.S. Pat. No. 6,725,866; “Current methods to treatgastroesophageal reflux disease consist of lifestyle changes such asweight loss, avoidance of certain foods that exacerbate the symptoms ofGERD and avoidance of excessive bending. Elevation of the head of thebed helps prevent nocturnal reflux. While these avoidance strategies maybe helpful, there is relatively little data supporting the efficacy oflifestyle modification alone for the treatment of GERD. Medications forthe treatment of GERD have been administered for years with little or nosuccess.” One symptom of GERD mentioned in the above referenced patentis dyspepsia. “Dyspepsia, or heartburn, is defined as a burningsensation or discomfort behind the breastbone or sternum and is the mostcommon symptom of GERD. Other symptoms of GERD include dysphasia,odynophagia, hemorrhage, water brash, and pulmonary manifestations suchas asthma, coughing or intermittent wheezing due to acid aspiration.Dyspepsia may also mimic the symptoms of a myocardial infarction orsevere angina pectoris.” GERD has also been linked to irregularities inthe upper airways, such as asthma. It is believed that the low levels ofacid released due to GERD are responsible for these irregularities. Itis often difficult to properly diagnose GERD, as many other conditionsmimic the symptoms of GERD. As is taught in U.S. Pat. No. 5,951,468“since the symptoms of GERD often mimic cardiac chest pain, thephysician must confirm that the symptoms are in fact due to reflux andnot to a cardiac condition.”

U.S. Pat. No. 4,168,703 teaches an alternative testing conditionsinvolve the monitoring the pressure within the lower esophagus. Thistesting procedure is deficient due to the substantial degree of patientdiscomfort involved with this method.

U.S. Pat. Nos. 4,503,859 and 5,117,827 teach a procedure to test forreflux which includes esophageal pH monitoring, with esophagealmanometry testing. This testing occurs by passing a catheter into theesophagus, and then monitoring pH and sphincter pressures for 24 hoursthrough the use of an ambulatory monitoring device. Test results arethen analyzed to determine reflux degree. Such ambulatory devices areextremely inconvenient for the patient. In addition, these testingconditions require a significant amount of time to obtain a result.

U.S. Pat. No. 5,254,591 discloses a pharmaceutical composition usefulfor treating gastroesophageal reflux disease. This patent clearly failsto disclose or suggest a method of detection for the disease.

U.S. Pat. No. 5,524,622 teaches a method of detecting increased bloodflow to certain regions of the gastrointestinal tract, and thus is ableto detect inflammation of the tract. This technique is deficient in thatit is significantly more complex and considerably more expensive thatthat of the instant invention. The equipment needed to conduct suchtesting is exceedingly specialized.

U.S. Pat. No. 5,571,116 discloses a minimally invasive device and methodfor the treatment of gastroesophageal reflux disease. The techniquestaught in this patent may be used in conjunction with the instantinvention. However, this patent fails to disclose a method of detectionof gastroesophageal reflux disease.

U.S. Pat. No. 5,738,110 teaches a retrievable capsule useful forobtaining samples of gastroesophegeal fluids. In this manner, epithelialcells may be removed for later testing. While this patent teachesmethods of obtaining samples for testing, it fails to teach testingmethodology itself.

U.S. Pat. No. 5,879,897 teaches an immunoassay technique for thedetection of pepsin. This patent provides “ . . . methods of detectingand diagnosing reflux diseases and disorders by detecting the presenceof pepsin in airway secretions (e.g. throat, lung, esophagus, or mouthmucus/sputum/saliva) or other bodily fluids of subjects suspected ofhaving a reflux disorder of disease. An advantage of the disclosedmethods over methods based on detection of pH changes is that pepsin orpepsinogen from reflux becomes trapped in the mucus and remains in thethroat or esophagus longer than acid (hydrogen ions) and can thus bedetected for hours or days after a reflux event.” The techniques taughtin U.S. Pat. No. 5,879,897 can be distinguished from the instantinvention by the nature of the detection system itself. The instantinvention, in one embodiment, detects the enzymatic activity of pepsin.The aforementioned antibody techniques are deficient in that they detectpepsin itself, even when such pepsin may not be an active enzyme.Additionally, such immunoassay techniques are known to take asignificant amount of time to obtain results.

U.S. Pat. No. 5,951,468 discloses a method for testing for esophagealacid sensitivity by attempting to induce the symptoms ofgastroesophageal reflux through the ingestion of weak acids. This patentis clearly distinguished from the instant invention by the very natureof the test.

U.S. Pat. No. 6,238,335 teaches a method and device for the treatment ofgastroesophageal reflux disease. The techniques taught in thisapplication involve the augmentation of the epithelial lining to promoteacid resistance.

U.S. Pat. No. 6,338,345 discloses a device and method for the deliveryof treatment agents into the esophagus for the treatment ofgastroesophageal reflux disease. Both of these patents fail to teach orsuggest a method of testing for gastroesophageal reflux disease.

U.S. Pat. No. 6,475,145 teaches a method for monitoring pH levels over aprolonged period of time. The techniques of this patent are deficient inthat they fail to teach or suggest a rapid testing method for thedetection of gastroesophageal reflux disease.

U.S. patent application 2004/0002168 discloses a test strip to test forcerebrospinal fluid. This application contains no suggestion ormotivation to modify this test strip to test for pepsin. Other teststrips and/or test strip reading devices are disclosed in U.S. Pat. No.6,180,063 (Measuring Device for Use with a Test Strip); U.S. Pat. No.6,315,951 (Test Strip Measuring System); U.S. Pat. No. 6,514,460(Luminous Glucose Monitoring Device); U.S. Pat. Nos. 6,514,461 and6,716,393 (System for Automatically Testing a Fluid Specimen); U.S. Pat.No. 6,689,320 (Test Strip Measuring System); and the like.

The content of U.S. Pat. Nos. 4,168,703; 4,455,381; 4,503,859;5,117,827; 5,254,591; 5,524,622; 5,571,116; 5,738,110; 5,879,897;5,951,468; 6,180,063; 6,238,335; 6,315,951; 6,338,345; 6,475,145;6,514,461; 6,689,320; 6,716,393; 6,725,866 and U.S. patent application2004/0002168 is hereby incorporated by reference into thisspecification.

Yet another testing method involves nasogastric intubation of thepatient and subsequently adding dilute acid to intentionally trigger thecondition to be tested for. As is known to those skilled in the art,this test is often referred to as the “Berstein acid perfusion test.”

As acknowledged in U.S. Pat. No. 5,951,468 “Currently, there is nosimple diagnostic test or procedure that can be done either in aphysician's office or in an individual's home to assist with thediagnosis of acid reflux and GERD.”

It is an object of this invention to provide at least one of thefollowing; a method for the detection of an enzyme that is rapid andconvenient enough that it can be performed in a physician's office orsimilar location without the need for ambulatory monitoring devices; amethod for detecting an enzyme wherein the enzyme can be detected inless than two hours (i.e. “point of care” detection).

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided an apparatusfor detecting pepsin comprising a solid support, and a peptide chainwherein the peptide chain is operatively configured to be cleaved bypepsin, and the peptide chain is disposed on a surface of the solidsupport.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by reference to the following drawings,in which like numerals refer to like elements, and in which:

FIG. 1 is a schematic diagram of one test strip of the invention;

FIG. 1A is a schematic diagram of a test strip with a first tagattached;

FIG. 1B is a schematic diagram of a test strip with the first tag ofFIG. 1A released;

FIG. 2 is a flow diagram of one process of the invention utilizing tags;

FIG. 3 is a schematic diagram of another test strip of the invention;

FIG. 3A is a schematic diagram of a test strip before enzymaticcleavage;

FIG. 3B is a schematic diagram of a test strip after enzymatic cleavage;

FIG. 4 is a flow diagram of one process of the invention utilizingenzymatic cleavage;

FIG. 5 is a flow diagram of one process of the invention utilizingenzymatic cleavage;

FIG. 6 is a flow diagram of another process of the invention utilizingenzymatic cleavage;

FIG. 7 is a schematic diagram of three suitable methods for obtainingsamples for use with the instant invention;

FIG. 7A is a schematic diagram of one method of tubation to obtainsamples;

FIG. 7B is a schematic diagram of one method of oral swabbing to obtainsamples; and

FIG. 7C is a schematic diagram of one method of passive samplecollection.

The present invention will be described in connection with a preferredembodiment, however, it will be understood that there is no intent tolimit the invention to the embodiment described. On the contrary, theintent is to cover all alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby the appended claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 illustrates one embodiment of the invention wherein pepsin isdetected in a sample (for example, in a human saliva sample) usingchemical tags. In one embodiment, a test strip 102, a substrate, a solidsupport, a stationary phase, or other immobilizing agent is used. In theembodiment shown in FIG. 1, test strip 102 is comprised of testing area104. Disposed on the surface of testing area 104 is a multiplicity ofpeptide chain 106. In one embodiment, peptide chain 106 contains anaromatic amino acid residue such as phenylalanine, tyrosine, tryptophan,histidine, and the like, which is operatively configured to be cleavedby enzymatic hydrolysis of the peptide bond by pepsin. A first end 106′of peptide chain 106 is attached to the testing area 104. A second end106″ of peptide chain 106 is attached to a first tag 108. Upon enzymaticcleavage of peptide chain 106 by pepsin (forming cleaved peptide chain107), first tag 108 is released to form released first tag 109. Thus,the concentration of released first tag 109 is diagnostic of thepresence of pepsin.

Suitable peptide chains include (from N to C terminus), but are notlimited to:-glycine-glycine-glycine-asparagine-X-asparagine-glycine-glycine-glycine - - -TAG where X is an aromatic amino acid (such as, for example,phenylalanine, tyrosine, tryptophan, and the like).

In one embodiment, it is preferred that the amino acid positionsflanking the aromatic amino acid X not be valine, alanine or glycine, asthe presence of such residues is known to diminish the activity ofpepsin. Suitable peptides can be obtained commercially, synthesized on acommercially available peptide synthesizer (in house) or synthesized byhand using standard solid phase chemical techniques.

In one embodiment, the test does not utilize a traditional test strip.Instead, a different solid support or other substrate is used. In oneembodiment of the present invention, the peptide or protein (containingour dye or indicator-tag) is covalently attached to the wall of anEppendorf tube or test tube or other similar container. The resultingsolution is then screened (following exposure to the solution that mayor may not contain pepsin obtained from the patient) for the release ofthe tag or dye, indicating the presence of pepsin.

In the embodiment shown in FIG. 2, a testing solution is used tovisualize the release of the first tag. In step 202 of the process 200,a sample to be tested is exposed to a test strip. This test stripcomprises the aforementioned peptide chain and first tag. If pepsin ispresent, the then the first tag will be released. In step 204 of theprocess, the test strip is exposed to a test solution. This testsolution comprises a second tag which is operatively configured tointeract with the released first tag. In one embodiment, the testsolution is a buffered solution designed to promote the enzymaticcleavage of the peptide chain by pepsin (pH 1-3, for optimum pepsinactivity). In one embodiment, the test strip is disposed within the testsolution for a period of less than about 3 hours, and preferably forless than about 2 hours. In step 206 pepsin cleaves the peptide bond andreleases the first tag into the test solution, where it contacts thesecond tag. Although some degree of cleavage may occur prior to theintroduction of the test strip into the test solution, in the embodimentdepicted in FIG. 2 most of the cleavage occurs after exposure to thetest solution. In step 208 the released first tag interacts with thesecond tag present in the test solution. This interaction is detectablein a variety of means, depending on the nature of the first and secondtags. In one embodiment, the chemical combination of the first andsecond tag may be detected visually (i.e. a change in coloration of thesolution). In another embodiment, the color changes that take place areoutside of the visual range of human color perception, and spectroscopicinstruments are used to detect the change. In yet another embodiment,the color changes are within the visual spectrum of the human eye, butspectroscopic instruments are used so as to quantify the measurements.Numerous suitable instruments for test strip analysis are known to thoseskilled in the art. Reference may be had to U.S. Pat. Nos. 4,748,114;5,797,693; 6,441,898; 6,458,596; 6,525,549; and the like. The content ofU.S. Pat. Nos. 4,748,114; 5,797,693; 6,441,898; 6,458,596; and6,525,549; is hereby incorporated by reference into this specification.Thus it is now feasible to compare the result to a colorimetric gradientand to determine not only the presence of pepsin but also quantify howmuch is present in a given sample.

FIG. 3 illustrates one embodiment of the invention wherein pepsin isdetected in a sample (for example, in a human saliva sample) withoutusing chemical tags. In one embodiment, a test strip 302 or other solidsupport/immobilizing agent is used. In the embodiment shown in FIG. 3,test strip 302 is comprised of testing area 304. Disposed on the surfaceof testing area 304 is a multiplicity of peptide chains 306. In oneembodiment, peptide chain 306 contains amino acid residues operativelyconfigured to be cleaved by enzymatic hydrolysis of the peptide bond bypepsin. A first end of peptide chain 306 is attached to the testing area304. Upon enzymatic cleavage of peptide chain 306 by pepsin, two peptidefragments are generated; cleaved peptide chain 307 and released peptidechain 309. This cleavage results in a new exposed terminus of cleavedpeptide chain 307. A dye may then be used that stains the chemicalfunctional group that has been exposed on the terminus of cleavedpeptide chain 307. Thus, the presence of the functional group on thecleaved peptide chain 307 is diagnostic of the presence of pepsin.

In the embodiment shown in FIG. 4, a series of solutions are used tovisualize the alteration of the peptide terminus. In step 402 of theprocess 400, a sample to be tested is exposed to a test strip. This teststrip comprises a peptide chain operatively configured to be cleaved bypepsin. If pepsin is present, then the peptide chain will be cleaved. Instep 404 of the process the test strip is exposed to a test solution. Inone embodiment, the test solution is a buffered solution designed topromote the enzymatic cleavage of the peptide chain by pepsin (pH 1-3,for optimum pepsin activity). In one embodiment, the test strip isdisposed within the test solution for a period of less than about 3hours, and preferably for less than about 2 hours. In anotherembodiment, the test strip is disposed within the test solution for aperiod of less than about 1 hour. In step 406, pepsin cleaves thepeptide bond and exposes a new peptide terminus. Although some degree ofcleavage may occur prior to the introduction of the test strip into thetest solution, in the embodiment depicted in FIG. 4 most of the cleavageoccurs after exposure to the test solution. In step 408 the cleavedpeptide chain is exposed to conditions that allow for the visualizationof the altered terminus. As would be appreciated by those skilled in theart, such staining techniques are well known. By way of illustration andnot limitation, one may use a solution of Coomassie Blue stain. Such asolution is known to stain primary amines on peptide chains. Othersuitable stains are commonly used on chromatography arts. Spectroscopictechniques similar to those discussed above can be used to detect subtlecolor changes. Such spectroscopic techniques can also be used to detectspectral changes outside of the visible spectrum.

In the embodiment shown in FIG. 5, a series of solutions are used tovisualize the alteration of the peptide terminus. The processillustrated in FIG. 5 is similar to that depicted in FIG. 4 except thatthe sample to be tested is disposed within the solution, and not placeddirectly on the test strip. In step 502 of the process 500, testsolution is prepared. This test solution is comprised of the sample tobe tested. In one embodiment, the test solution is further comprised ofbuffers to enhance the rate of enzymatic cleavage. In one embodiment,the test solution is a buffered solution designed to promote theenzymatic cleavage of the peptide chain by pepsin (pH 1-3, for optimumpepsin activity). In step 504 of the process the test strip is exposedto the test solution. This test strip comprises a peptide chainoperatively configured to be cleaved by pepsin. If pepsin is present inthe test solution, the then peptide chain will be cleaved. In oneembodiment, the test strip is disposed within the test solution for aperiod of less than about 3 hours, and preferably for less than about 2hours. In step 506, pepsin cleaves the peptide bond and exposes analtered terminus. Although some degree of cleavage may occur prior tothe introduction of the test strip into the test solution, in theembodiment depicted in FIG. 5 most of the cleavage occurs after exposureto the test solution. In step 508 the cleaved peptide chain is exposedto conditions that allow for the visualization of the altered terminus.By way of illustration and not limitation, one may use a solution ofCoomassie Blue stain. Such a solution is known to stain primary amineson peptide chains. Spectroscopic techniques similar to those discussedabove can be used to detect subtle color changes. Such spectroscopictechniques can also be used to detect spectral changes outside of thevisible spectrum.

In another embodiment shown in FIG. 6, small proteins themselves areutilized as substrates of pepsin (bovine serum albumen for example).Reference may be had to process 600 depicted in FIG. 6. In theembodiment depicted in FIG. 6, protein 602 contains many pepsin cleavagesites. In step 608 of the process 600, protein 602 is labeled using tags604. In step 610, labeled protein 603 is exposed to a solution whichcontains active pepsin. Active pepsin then cleaves labeled protein 603which produces protein fragments 606, complete with liberated tags 612.The analytical techniques discussed elsewhere in this specification arethen utilized to detect protein fragments 606. The concentration ofpepsin is then related to the number of protein fragments 606, ascompared to the number of labeled proteins 603 which were uncleaved.

Suitable tags include any compound which undergoes a detectable chemicalchange. For example, azo compounds are easily formed and producebrightly colored compounds. Typical reactions involve utilizingcommercially available diazonium salts and various substituted phenolsor naphthols. The range of colors generated from such reactions oftenvary from yellow to orange to red to purple depending on thefunctionality and substitution patterns of both the diazonium and thephenol/naphthol.

Aromatic alcohols are known to undergo reactions with diazonium salts toform colorful azo compounds. For example, naphthol reacts with Fast Red(a commercially available diazonium salt) to generate a bright redcolor.

In one embodiment of the invention, this technique is adapted to detectactive pepsin. In one such embodiment, the phenol or naphthol isattached to the terminus of a desired amino acid sequence containingaromatic residues. In the presence of pepsin the amide bond of thearomatic amino acid is then hydrolyzed and the arene is released fromthe solid support and thereafter resides in solution. Reference may behad to equation 3. A diazonium salt is then added to the solution andthe salt and phenol/naphthol react and generate the colorful azocompound.

As will be appreciated by those skilled in the art, phenols are aromaticalcohols that have a single aromatic ring. Naphthols are aromaticalcohols which have two aromatic rings. Diazonium salts are compounds ofthe general formula RN₂X where X is an anion. It is preferred to use anaromatic diazonium salt in conjunction with the instant invention. Theanion X may be a halide (Fast Red), an organic sulfoxide (Fast Red B),and the like.

The proteins or peptides discussed in this application could be easilyattached to a strip, solid support or other substrate via standardchemical techniques. In one embodiment an imine is formed at theN-terminal domain of either a protein or peptide using an aldehydefunctionalized support. In another embodiment an imine is formed usingglutaric dialdehyde in conjunction with an amine functionalized support.One skilled in the art will appreciate that some imine formations arereversible under certain conditions. A variety of techniques have beendeveloped to prevent such reversible reactions. For example, one suchtechnique involes the further modification of the imine via chemicalreduction (using sodium cyanoborohydride or sodium borohydride, etc) toyield amines, which are non-reversible products.

In another embodiment, the attachment to the support is via activationof carboxylic acids (either C-terminal acid group or acid groupscontained within the peptide or protein side chains). Carboxylic acidscan be activated via many different methods, but the most common (and“protein friendly”) are activation with DIC (diisopropylcarbodiimide),DCC (dicyclohexylcarbodiimide), DMAP (dimethylaminopyridine), HOBt(hydroxybenzotriazole), to name a few. Attachment using this methodwould be facilitated via an amine functionalized support.

In yet another embodiment, attachment to the support is achieved usingCDI (carbonyl diimidazole). This reacts with free amines on thesubstrate followed by exposure to the protein or peptide. The activatedsupport is then treated with free amines contained within the substrateto be immobilized; either a protein or peptide.

It will be apparent to one skilled in the art that a variety of othersuitable techniques for the attachment of proteins or peptides to solidsurfaces are known. The aforementioned examples are intended to beillustrative, and not limiting.

As illustrated in FIG. 7, samples may be obtained by conventional means.For example, samples may be obtained passively (i.e. the patientproduces the sample). For example, saliva may be obtained, vigorouscoughing may provide sputum (see FIG. 7C), or esophageal secretion maybe obtained. Reference may be had to U.S. Pat. No. 5,372,126 to Blau(Pulmonary Sampling Chamber); U.S. patent application 2004/0029205 toSmall (Diagnostic System for Differentiating Sputum from Saliva), andthe like. Samples may also be obtained actively (i.e. medical personalassists in obtaining the sample). In one embodiment, the oral cavity isswabbed to obtain a sample (see FIG. 7B). Reference may be had to U.S.Pat. No. 4,795,421 to Blasius (Oral Hygiene Swab); U.S. Pat. No.5,056,521 to Parsons (Method for Monitoring Glucose Level); U.S. Pat.No. 5,975,897 to Propp (Oral Suctioning Swab); and the like. In anotherembodiment, a device, such as a catheter is passed into the esophagus orupper airway through the nose or mouth to collect the sample (see FIG.7A). In one such embodiment, a nasoesophageal tube is used to collectthe esophageal specimen. In another embodiment, a catheter is attachedto, or passed along side of an endoscope (i.e. bronchoscope) to collectthe sample. In yet another embodiment, the sample is collected throughroutine esophagoscopy or bronchoscopy (i.e. by suctioning the sample outof the lung or esophagus). Reference may be had to U.S. Pat. No.4,880,015 to Nierman (Biopsy Forceps); U.S. Pat. No. 4,702,260 to Wang(Flexible Bronchoscopic Needle Assembly); and the like. The content ofU.S. Pat. Nos. 4,702,260; 4,795,421; 4,880,015; 5,056,521; 5,372,126;5,975,897; and U.S. patent application 2004/0029205 is herebyincorporated by reference into this specification. Many other suitabletechniques would likewise be apparent to one skilled in the art.

As used in this specification, the term “solid support” refers to animmobile support such as, for example, a test strip, a wall of a testtube or similar container, an Eppendorf tube, and the like. A variety ofsuitable solid supports are readily available. Reference may be had toU.S. Pat. Nos. 4,748,114; 5,797,693; 6,441,898; 6,458,596; 6,525,549;U.S. patent application 2004/0002168 and the like. The content of eachof these patents is hereby incorporated by reference into thisspecification. In one embodiment, the test strip is rectangular. In oneembodiment, the test strip is comprised of cellulose fibers. In anotherembodiment, the test strip is cylindrical.

As used in this specification, the term “peptide chain” refers to achain of amino acids, peptides, proteins, or similar biomolecules, whichcan be cleaved by an enzyme, and pepsin in particular. Suitable peptideschains are known to those skilled in the art.

As used in this specification, the term “first tag” refers to a chemicalmoiety placed at one terminus of a peptide chain for later reaction witha second tag. Suitable first tags are likewise known in the art.Examples of suitable first tags include aromatic alcohols, such assubstituted phenols, naphthols, and the like.

As used in this specification, the term “chemical dye” refers to acompound capable of staining severed peptide chains. Suitable compoundsinclude, for example, Coomassie brilliant blue, Texas Red, pyrene,fluoroscein, and the like) that is in conjugation with a reactive group,usually an amine. In one such embodiment the dye contains an aldehyde,for example, or an activated ester (as described previously) and isattached via a free amine present on the peptide or protein. Theaforementioned dyes are readily available from commercial sources(Aldrich Chemical) and receive wide usage in chromatography arts.

As used in this specification, the term “second tag” refers to achemical moiety which undergoes a chemical reaction with a first tag togenerate a detectable complex. Suitable second tags are also available.In one embodiment, a second tag undergoes a chemical reaction with thecleaved peptide chain, rather than the first tag (i.e. a peptide stainsuch as Coomassie Blue). Examples include the aforementioned azocompounds.

As used in this specification, the term “antibody” refers to anyimmunoglobulin that binds specifically to an antigenic determinant. Theterm “non-antibody” therefore refers to a material which is not anantibody. Reference may be had to U.S. patent application 2004/0002168.

It is, therefore, apparent that there has been provided, in accordancewith the present invention, a method and apparatus for the detection ofpepsin. While this invention has been described in conjunction withpreferred embodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Alternative enzymes, other than pepsin, are clearly detectableusing the techniques taught in the instant disclosure. Accordingly, itis intended to embrace all such alternatives, modifications andvariations that fall within the spirit and broad scope of the appendedclaims.

1. An article for detecting the presence of pepsin suitable to test asubject in a physician's office, the subject suspected of suffering fromgastroesophageal reflux disease and providing a sample suspected ofcontaining pepsin, the article comprising: a. a solid support comprisinga test area surface; b. a multiplicity of peptide chains each having apeptide proximal end and a peptide distal end, wherein the peptidedistal end comprises a peptide bearing a first tag that interacts with asecond tag to provide a detectable chromophoric product, wherein all ofsaid peptide proximal ends are attached to said test area surface ofsaid support, and wherein said peptide chains are cleavable by saidpepsin to provide said peptide proximal end attached to said test areasurface and said peptide distal end bearing the detectable first tagcleaved from said test area surface.
 2. The article as recited in claim1, wherein said solid support is a test strip.
 3. The article as recitedin claim 2, wherein said test strip comprises a distal end and aproximal end, and wherein said test area surface is disposed proximateto said distal end.
 4. The article as recited in claim 1, wherein saidsolid support is a tube.
 5. The article as recited in claim 4, whereinsaid tube is a glass tube.
 6. The article as recited in claim 4, whereinsaid tube is an Eppendorf tube.
 7. The article as recited in claim 1,wherein said detectable first tag comprises an aromatic alcohol.
 8. Thearticle as recited in claim 1, wherein each of said peptide chainscomprises an aromatic amino acid.
 9. The article as recited in claim 1,wherein said peptide chains comprise an amino acid residue joined by apeptide bond cleavable by said pepsin.
 10. The article as recited inclaim 9 wherein said amino acid residue is selected from the groupconsisting of phenylalanine, tyrosine, tryptophan, and histidine. 11.The article described in claim 10 wherein said amino acid residue ispreceded by an asparagine residue and followed by an asparagine residue.12. The article described in claim 1 wherein the peptide chain comprisesthe sequence —Y—X—Y—, wherein each Y is independently any amino acidresidue other than valine, alanine, or glycine, and X is an aromaticamino acid.
 13. The article described in claim 12 wherein X is selectedfrom the group consisting of phenylalanine, tyrosine, tryptophan, andhistidine.
 14. The article described in claim 12 wherein each Y isasparagine.
 15. The article described in claim 1 wherein the articledetects the presence of pepsin in about 2 hours or less.
 16. The articledescribed in claim 1 wherein the second tag comprises an aromaticdiazonium salt.
 17. The article described in claim 1 wherein thechromophoric product is detectable by the human eye, thereby permittingvisual detection of pepsin.
 18. The article described in claim 1 furthercomprising means for obtaining said sample of material.
 19. The articledescribed in claim 18 wherein said means for obtaining said sample ofmaterial comprises a catheter.
 20. An apparatus for detecting thepresence of pepsin in a sample of material suitable to test a subject ina physician's office, the subject suspected of suffering fromgastroesophageal reflux disease and providing the sample, said apparatuscomprising: a. an article for exposure to said sample of material, saidarticle comprising a substrate having a test area surface and amultiplicity of peptide chains each having a peptide proximal end and apeptide distal end, wherein the peptide distal end comprises a peptidebearing a detectable first tag, wherein all of said peptide proximalends are attached to said test area surface of said substrate, andwherein said peptide chains are cleavable by said pepsin to provide saidpeptide proximal end attached to said test area surface and said peptidedistal end bearing the detectable first tag cleaved from said test areasurface; and b. a container comprising a fillable volume for receiving atest solution comprising at least one of the sample, a buffer and asecond tag that interacts with the first tag to provide a detectablechromophoric product, and wherein at least the test area of said articleis insertable in said fillable volume.
 21. The apparatus as recited inclaim 20, wherein said substrate and said container are unitary.
 22. Theapparatus as recited in claim 21, wherein said container is a tube. 23.The apparatus as recited in claim 22, wherein said tube is a glass tubeor an Eppendorf tube.
 24. The apparatus as recited in claim 20, whereinsaid substrate is a test strip.
 25. The apparatus as recited in claim24, wherein said test strip comprises a strip distal end and a stripproximal end, and wherein said test area surface is disposed proximateto said distal end.
 26. The apparatus as recited in claim 24, whereinsaid test strip comprises cellulose fibers.
 27. The apparatus as recitedin claim 20, wherein said detectable first tag comprises an aromaticalcohol.
 28. The apparatus as recited in claim 20, further comprisingthe test solution disposed in the fillable volume.
 29. The apparatus asrecited in claim 28, wherein said test solution comprises the secondtag.
 30. The apparatus as recited in claim 29, wherein said second tagis a chemical dye.
 31. The apparatus as recited in claim 28, whereinsaid test solution comprises a buffer.
 32. The apparatus as recited inclaim 28, wherein said test solution comprises an aromatic diazoniumsalt.
 33. The apparatus as recited in claim 20, further comprising atest solution disposed in said fillable volume of said container. 34.The apparatus as recited in claim 20, further comprising means forobtaining said sample of material.
 35. The apparatus as recited in claim34, wherein said means for obtaining said sample of material comprises acatheter.
 36. The apparatus described in claim 20 wherein the peptidechain comprises the sequence —Y—X—Y—, wherein each Y is independentlyany amino acid residue other than valine, alanine, or glycine, and X isan aromatic amino acid.
 37. The apparatus described in claim 36 whereinX is selected from the group consisting of phenylalanine, tyrosine,tryptophan, and histidine.
 38. The apparatus described in claim 36wherein each Y is asparagine.
 39. The apparatus described in claim 20wherein the apparatus detects the presence of pepsin in a sample ofmaterial in about 2 hours or less.
 40. The apparatus described in claim20 wherein the chromophoric product is detectable by the human eye,thereby permitting visual detection of pepsin.
 41. The apparatusdescribed in claim 20 wherein each of said peptide chains comprises anaromatic amino acid.
 42. The apparatus described in claim 20 whereinsaid peptide chains comprise an amino acid residue joined by a peptidebond cleavable by said pepsin.
 43. The apparatus described in claim 42wherein said amino acid residue is selected from the group consisting ofphenylalanine, tyrosine, tryptophan, and histidine.
 44. The apparatusdescribed in claim 43 wherein said amino acid residue is preceded by anasparagine residue and followed by an asparagine residue.